Pharmacokinetics and competitive pharmacodynamics of ADP-induced platelet activation after oral administration of clopidogrel to horses

Jeffrey W. Norris 1Department of Pharmacology, College of Graduate Studies, Midwestern University, Glendale, AZ 85308.

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Johanna L. Watson 2Department of Medicine and Epidemiology, School of Veterinary Medicine, University of California-Davis, Davis, CA 95616.

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Fern Tablin 3Department of Anatomy, Physiology, and Cell Biology, School of Veterinary Medicine, University of California-Davis, Davis, CA 95616.

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Tania A. Kozikowski 5Idexx Laboratories Inc, 1 Idexx Dr, Westbrook, ME 04092.

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Heather K. Knych 4Department of Veterinary Molecular Biosciences and the K. L. Maddy Equine Analytical Chemistry Laboratory, School of Veterinary Medicine, University of California-Davis, Davis, CA 95616.

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Abstract

OBJECTIVE

To determine pharmacokinetics and pharmacodynamics after oral administration of a single dose of clopidogrel to horses.

ANIMALS

6 healthy adult horses.

PROCEDURES

Blood samples were collected before and at various times up to 24 hours after oral administration of clopidogrel (2 mg/kg). Reactivity of platelets from each blood sample was determined by optical aggregometry and phosphorylation of vasodilator-stimulated phosphoprotein (VASP). Concentrations of clopidogrel and the clopidogrel active metabolite derivative (CAMD) were measured in each blood sample by use of liquid chromatography–tandem mass spectrometry, and pharmacokinetic parameters were determined with a noncompartmental model.

RESULTS

Compared with results for preadministration samples, platelet aggregation in response to 12.5μM ADP decreased significantly within 4 hours after clopidogrel administration for 5 of 6 horses. After 24 hours, platelet aggregation was identical to that measured before administration. Platelet aggregation in response to 25μM ADP was identical between samples obtained before and after administration. Phosphorylation of VASP in response to ADP (20μM) and prostaglandin E1 (3.3μM) was also unchanged by administration of clopidogrel. Time to maximum concentration of clopidogrel and CAMD was 0.54 and 0.71 hours, respectively, and calculated terminal-phase half-life of clopidogrel and CAMD was 1.81 and 0.97 hours, respectively.

CONCLUSIONS AND CLINICAL RELEVANCE

Clopidogrel or CAMD caused competitive inhibition of ADP-induced platelet aggregation during the first 24 hours after clopidogrel administration. Because CAMD was rapidly eliminated from horses, clopidogrel administration may be needed more frequently than in other species in which clopidogrel causes irreversible platelet inhibition. (Am J Vet Res 2019;80:505–512)

Abstract

OBJECTIVE

To determine pharmacokinetics and pharmacodynamics after oral administration of a single dose of clopidogrel to horses.

ANIMALS

6 healthy adult horses.

PROCEDURES

Blood samples were collected before and at various times up to 24 hours after oral administration of clopidogrel (2 mg/kg). Reactivity of platelets from each blood sample was determined by optical aggregometry and phosphorylation of vasodilator-stimulated phosphoprotein (VASP). Concentrations of clopidogrel and the clopidogrel active metabolite derivative (CAMD) were measured in each blood sample by use of liquid chromatography–tandem mass spectrometry, and pharmacokinetic parameters were determined with a noncompartmental model.

RESULTS

Compared with results for preadministration samples, platelet aggregation in response to 12.5μM ADP decreased significantly within 4 hours after clopidogrel administration for 5 of 6 horses. After 24 hours, platelet aggregation was identical to that measured before administration. Platelet aggregation in response to 25μM ADP was identical between samples obtained before and after administration. Phosphorylation of VASP in response to ADP (20μM) and prostaglandin E1 (3.3μM) was also unchanged by administration of clopidogrel. Time to maximum concentration of clopidogrel and CAMD was 0.54 and 0.71 hours, respectively, and calculated terminal-phase half-life of clopidogrel and CAMD was 1.81 and 0.97 hours, respectively.

CONCLUSIONS AND CLINICAL RELEVANCE

Clopidogrel or CAMD caused competitive inhibition of ADP-induced platelet aggregation during the first 24 hours after clopidogrel administration. Because CAMD was rapidly eliminated from horses, clopidogrel administration may be needed more frequently than in other species in which clopidogrel causes irreversible platelet inhibition. (Am J Vet Res 2019;80:505–512)

Local and systemic inflammation is recognized as contributing to the development of a prothrombotic environment in the microvasculature.1 For several diseases of horses, including acute laminitis and limb cellulitis, the formation of thrombi contributes to the disease process.2,3 In addition to localized vascular thrombosis, persistent laminitis is a common sequela of cellulitis of the limbs.3,4 In horses in which laminitis has been experimentally induced by the administration of oligofructose, platelet activation has preceded the onset of lameness.5

One measure of platelet activation in experimentally induced acute laminitis is an increase in circulating concentrations of serotonin, which is detectable in the interval between oligofructose administration and the onset of lameness.6 Serotonin is stored in a subset of the secretory granule population of platelets termed dense granules, and release of serotonin is consistent with the development of a prothrombotic microenvironment. These dense granules also contain the platelet agonist ADP, which can act in an autocrine manner and has a potency 12-fold as great as that of serotonin for inducing aggregation of equine platelets.7 Therefore, inhibition of ADP-induced platelet activation may be a useful adjunct treatment to improve outcomes in equine patients with diseases that produce such prothrombotic environments.

Clopidogrel is approved for prophylactic use to reduce the risk of thrombosis in myocardial infarction and stroke in humans.8 Clopidogrel also reduces ADP-induced platelet activation in cats with hypertrophic cardiomyopathy and improves survival of cats at risk of developing thromboemboli.9,10 In these species as well as dogs, clopidogrel rapidly and irreversibly inhibits ADP-induced platelet activation.9–12 The therapeutic efficacy of clopidogrel for the treatment of horses with diseases that produce a prothrombotic environment has not been reported. Although irreversible inhibition of ADP-induced aggregation of equine platelets has been attained after treatment with clopidogrel for 2 days, inhibition is typically submaximal and in some cases can diminish within the first 24 hours following treatment.13,14 Additionally, the pharmacokinetics of clopidogrel in horses has not been completely described.

Therefore, the purpose of the study reported here was to determine the pharmacokinetics of clopidogrel in horses and to assess effects of clopidogrel on ADP-induced aggregation of equine platelets. We hypothesized that there would be reversible inhibition of ADP-induced aggregation of equine platelets for the first 24 hours after administration of clopidogrel.

Materials and Methods

Animals

Six healthy horses housed at the Center for Equine Health of the University of California-Davis were used in the study. Horses comprised 4 mares and 2 geldings and were between 8 and 18 years of age. There were 2 Quarter Horses, 2 Thoroughbreds, and 2 Standardbreds. All procedures were conducted in accordance with an institutionally approved protocol.

Procedures

Food was withheld from the horses overnight prior to administration of clopidogrel. The next morning, the horses were weighed, and vital signs (body temperature, respiratory rate, and heart rate) were obtained. To measure gastric pH and determine the association with clopidogrel absorption (pKa = 4.55), a nasogastric tube was passed, and 1 L of gastric reflux was collected. A blood sample (8.5 mL) was collected into an evacuated tube containing citratea by venipuncture of an external jugular vein; all subsequent blood samples were collected in the same manner. Clopidogrel tabletsb (75 mg/tablet) were crushed and dissolved in 50 mL of lemon juice. Lemon juice was used as the vehicle for the drug because it naturally contains 5% to 6% citric acid, which would shift the equilibrium of solubilized clopidogrel to the protonated state. Clopidogrel (2 mg/kg) was administered via the nasogastric tube within 10 minutes after preparation. The dose administered to each horse was rounded to the nearest value for whole tablets (ie, 75 mg).

PRP preparation and optical aggregometry

Within 60 minutes after blood samples were collected, they were placed in 17 × 120-mm conical polypropylene tubes and centrifuged (200 × g for 15 minutes at room temperature [approx 25°C]). The platelet count in the resulting PRP was measured,c adjusted to 300,000 ± 25,000 cells/μL, and maintained at 37°C until aggregometry experiments were performed. Platelet aggregation was measuredd for up to 16 minutes after baseline optical density was established, and ADPe was added at doses of 12.5 or 25μM.

Platelet preparation and western blotting

An aliquot (500 μL) of PRP was transferred to a microcentrifuge tube, and apyrase and EDTA (final concentrations, 0.1 U/mL and 1mM, respectively) were added. Tubes were then centrifuged at 3,000 × g for 2 minutes at room temperature. After centrifugation was completed, platelet-poor plasma was removed, and the platelet pellet was suspended in 500 μL of Tyrode-HEPES (12mM NaHCO3, 138mM NaCl, 2.9mM KCl, and 10mM 4-[2-hydroxyethyl]-1-piperazineethanesulfonic acid; pH, 7.2). Washed platelets were divided into 150-μL aliquots containing equal numbers of platelets and incubated with 3.3μM PGE1,f 20μM ADP, or the combination of 3.3μM PGE1 and 20μM ADP for 5 minutes at 37°C.15 Reactions were terminated and prepared for western blot analysis by the addition of protease inhibitorsg (final concentrations, 4mM 4-[2-aminoethyl]benzenesulfonyl fluoride hydrochloride, 1μM leupeptin, 1μM pepstatin, 0.3μM aprotinin, and 100 μg of soybean trypsin inhibitor/mL) and 2× Laemmli buffer (20% glycerol, 4% SDS, 20mM EDTA, 200mM dithiothreitol,h and 50mM tris[hydroxymethyl]aminomethane; pH, 6.8). Lysates were resolved by electrophoresisi through 7% polyacrylamide gels containing 1% SDS and transferred to nitrocellulose membranes, which were incubated with anti–phosphorylated-VASPj and horseradish peroxidase–conjugated goat antimousek secondary antibodies. Chemiluminescence resulting from incubation with substratel was captured with a digital imager and quantified.m

Purification and mass spectrometry of clopidogrel and CAMD

Within 10 minutes after blood was collected, 2-bro-mo-3′ methoxyacetophenonen (final concentration, 2.5mM) was added to 1 mL of blood to inactivate the reactive thiol group of the clopidogrel active metabolite.16 Samples then were stored at −80°C until analysis.

Stock solutions were diluted with methanol to create working solutions with concentrations of 0.01, 0.1, 1, and 10 μg/mL. Plasma calibrators were prepared by dilution of the working solutions with drug-free equine plasma to achieve concentrations ranging from 0.25 to 12 ng/mL. Calibration curves and negative control samples were prepared fresh for each quantitative assay. In addition, quality control samples (equine plasma fortified with clopidogrel at concentrations of 0.8, 4.5, and 11.0 ng/mL) were included with each sample set as an additional evaluation of accuracy.

Before samples were analyzed, 0.2 mL of plasma was diluted with 0.2 mL of acetonitrile:1M acetic acid (9:1 [vol:vol]) containing d4-clopidogrelo (20 ng/mL) to precipitate proteins. Samples were mixed on a vortex device for 1.5 minutes, refrigerated for 30 minutes, mixed on the vortex device for an additional 0.3 minutes, and centrifuged at 3,830 × g for 10 minutes at 4°C. Then, 15 μL was injected into a liquid chromatography–tandem mass spectrometry system, and concentrations of clopidogrel and CAMD were measured. Quantitative analysis of plasma was performed on a mass spectrometerp coupled with an ultrahigh-pressure liquid chromatography system.q The system was operated in positive electrospray ionization at a resolution of 60,000 full width at half maximum. Spray voltage was set at 4,300 V; sheath gas and auxiliary gas were 40 and 10 arbitrary units, respectively; and capillary temperature was 350°C. Product masses and collision energies were optimized by infusion of the standard solutions into the mass spectrometer. Chromatography was performed with a C18 columnr and a linear gradient of acetonitrile in water with 0.2% formic acid. Flow rate was 0.3 mL/min. The initial acetonitrile concentration was held at 3% for 0.5 minutes, increased to 70% over 7.5 minutes, increased to 90% over 1.0 minute, and held at that concentration for 0.5 minutes, before re-equilibration for 3.5 minutes at initial conditions.

Detection and quantification were conducted by use of a full-scan tandem mass spectrometry accurate mass ion trap of initial precursor ion for clopidogrel (m/z, 322.1), CAMD (m/z, 504.1), and the internal standard d4-clopidogrel (m/z, 326.1). All tandem mass spectrometry transitions had an isolation width of 1.5 and collision energy of 35 V. Response for the 2 predominant product ions for clopidogrel (m/z, 184.08 and 212.05), CAMD (m/z, 324.10 and 354.06), and the internal standard d4-clopidogrel (m/z, 188.08 and 216.07) were plotted with a mass tolerance of 10 ppm, and peaks at the proper retention time were integrated with software.s The software was used to generate calibration curves and quantitate clopidogrel in all samples by use of linear regression analysis. A weighting factor of 1/X was used for all calibration curves. There was no CAMD standard available; therefore, CAMD concentrations were estimated by use of the clopidogrel calibration curve, which was generated with peak areas.

Intraday, interday, and analyst-to-analyst precision and accuracy of the assay were determined by assaying quality control samples in replicates (n = 6). Accuracy was reported as the percentage of the nominal concentration, and precision was reported as the percentage of the relative SD. The limit of quantitation was the lowest calibrator that could be measured with acceptable precision and accuracy, and the limit of detection was established on the basis of the lowest calibrator with a signal-to-noise ratio of 3:1.

Pharmacokinetic calculations

Noncompartmental analysis performed with commercially available softwaret was used for determination of pharmacokinetic parameters for clopidogrel and CAMD. Values for t1/2, AUC from time 0 to infinity, and extrapolated percentage of the AUC were determined. The t1/2 was calculated as 0.693 divided by the terminal slope, and AUC was calculated with the logarithmic up–linear down trapezoidal method and extrapolated to infinity by use of the last measured plasma concentration divided by the terminal slope.

Statistical and pharmacokinetic analyses

Slopes, amplitudes, and lag times from aggregometry experiments were calculated by use of the manufacturer's software.d Platelet reactivity indices for western blotting were calculated from VASP phosphorylation experiments by use of quantified band intensities corresponding to treatment with PGE1 or ADP and PGE1 as follows17:

Platelet reactivity index for western blotting = (band intensity of samples treated with PGE1 – band intensity of samples treated with ADP and PGE1)/band intensity of samples treated with PGE1

For absorption minima determined in aggregometry experiments and platelet reactivity indices determined in VASP phosphorylation experiments, differences between samples collected before clopidogrel administration, compared with those collected after clopidogrel administration, were determined by use of ratio-paired, 2-tailed t tests. Values were considered significant at P < 0.05. Linear relationships between pharmacokinetic parameters and gastric pH or aggregation were determined by least squares fitting and were only considered significant for R2 > 0.8.

Results

The addition of 12.5μM ADP to PRP caused a decrease in optical absorption that corresponded to the progressive aggregation of platelets (Figure 1). In samples collected before administration of clopidogrel, absorption decreased to a minimum that did not return to the established baseline value over the course of the study, which indicated that aggregation was complete. This complete response of equine platelets to ADP diminished within 2 hours after administration of clopidogrel. This diminishment of response persisted for up to 12 hours, but platelets from horses administered clopidogrel regained the capacity to aggregate in response to ADP by 24 hours.

Figure 1—
Figure 1—

Aggregation response of equine platelets to ADP before (time 0) and after oral administration of a single dose of clopidogrel (2 mg/kg). A—Representative absorption curves for PRP in response to 12.5μM ADP. Samples were collected before (solid black line) and 2 (dashed black line), 4 (dotted black line), 6 (solid gray line), 12 (dashed gray line), and 24 (dotted gray line) hours after clopidogrel administration. Time of ADP addition is indicated (arrow). B—Minimum relative absorption of PRP after addition of 12.5μM ADP. Samples were obtained from 6 horses before and 12 hours after administration of clopidogrel. Each symbol represents values for 1 horse; the white symbols represent values for a horse with platelets that responded normally to ADP at all times after drug administration. C—Minimum relative absorption of PRP in response to 12.5μM ADP for all samples obtained from 5 horses in which platelet aggregation was inhibited by clopidogrel. Each symbol represents results for 1 horse, and the mean for all 5 horses at each time is indicated (horizontal line). *Platelet aggregation was significantly (P < 0.05) inhibited, compared with preadministration values. D—Comparison of minimum relative absorption for equine PRP in response to 12.5μM ADP (circles) or 25μM ADP (squares) before and after administration of clopidogrel. Values represent mean ± SD for 5 horses. ‡Within a time point, value differs significantly (P < 0.01) for stimulation with 12.5μM ADP from the value for simulation with 25μM ADP.

Citation: American Journal of Veterinary Research 80, 5; 10.2460/ajvr.80.5.505

For 5 horses of the study, the response to ADP for PRP obtained 12 hours after administration of clopidogrel was diminished by 18% to 42% relative to that measured before clopidogrel administration (Figure 1). However, for the other horse in the study, the response to ADP for PRP obtained 12 hours after administration of clopidogrel was diminished by only 3%, compared with that measured for the initial sample. Platelets from that horse had normal responses to ADP at all times after administration of clopidogrel.

For the 5 horses that responded to clopidogrel, platelet aggregation induced by 12.5μM ADP was significantly inhibited at all times from 4 to 12 hours after clopidogrel administration (Figure 1). At 24 hours after clopidogrel administration, the platelet response to 12.5μM ADP was identical to that measured before administration of clopidogrel. Platelets obtained from the responders also aggregated in response to 25μM ADP at all times after administration of clopidogrel, which was consistent with the hypothesis that clopidogrel is a reversible inhibitor of aggregation of equine platelets within the first 24 hours after administration.

The hypothesis was further tested by measurement of phosphorylation of the intracellular protein VASP, which was eliminated in response to treatment with 20μM ADP at all times throughout the study (Figure 2). The addition of PGE1, a physiologic inhibitor of the ADP receptor P2Y1, induced marked VASP phosphorylation in unstimulated platelets. Stimulation with a combination of PGE1 and 20μM ADP induced signaling through the ADP receptor P2Y12, the target of clopidogrel, and caused mild, transient VASP phosphorylation in platelets obtained from horses after administration of clopidogrel. For platelets of the 5 horses that responded to clopidogrel, VASP was not consistently phosphorylated in response to the combination of ADP and PGE1. The platelet reactivity index reflective of this phosphorylation was not significantly decreased at any time after clopidogrel administration, compared with preadministration values, which again was consistent with the hypothesis that clopidogrel was a reversible inhibitor of activation of equine platelets during the 24-hour time course.

Figure 2—
Figure 2—

Results for VASP phosphorylation of equine platelets after oral administration of a single dose of clopidogrel. A—Representative western blots of VASP phosphorylation in washed platelets obtained from horses before (time 0) and at various times after oral administration of clopidogrel. Platelets were incubated with 20μM ADP (lane A), 3.3μM PGE1 (lane P), or both 20μM ADP and 3.3μM PGE1 (lane B). Equal numbers of platelets were loaded in each lane. B—Mean ± SD values for the platelet reactivity index (PRI) determined from relative VASP phosphorylation for 5 horses with substantial inhibition of platelet aggregation in response to clopidogrel. The PRI is a variable without units.

Citation: American Journal of Veterinary Research 80, 5; 10.2460/ajvr.80.5.505

Plasma concentrations of clopidogrel were measured in horses after administration, and the measurement response for clopidogrel was linear and yielded correlation coefficients ≥ 0.99. Intraday, interday, and analyst-to-analyst precision and accuracy of the assay were reported (Table 1). Accuracy and precision for all matrices were considered acceptable on the basis of the US FDA guidelines for bioanalytical method development. The limit of quantitation was 0.25 ng/mL, and the limit of detection for clopidogrel was approximately 0.2 ng/mL. Clopidogrel and CAMD were measurable in the plasma of 5 horses for up to 6 hours after administration (Figure 3). Pharmacokinetic parameters for the noncompartmental model were determined for these 5 horses (Table 2).

Figure 3—
Figure 3—

Mean ± SD plasma concentrations of clopidogrel (circles and solid line) and CAMD (squares and dashed line) before (time 0) and after oral administration of a single dose of clopidogrel to horses (n = 5; A), and peak plasma concentrations of clopidogrel (circles) and CAMD (squares) as a function of gastric pH in 6 horses (B). Clopidogrel and CAMD concentrations were estimated on the basis of the clopidogrel calibration curve. In panel A, time until maximum plasma concentrations of clopidogrel and CAMD was detected ranged from 15 to 45 minutes and 30 to 60 minutes, respectively.

Citation: American Journal of Veterinary Research 80, 5; 10.2460/ajvr.80.5.505

Table 1—

Accuracy and precision for analysis of clopidogrel concentration in equine plasma by use of liquid chromatography–tandem mass spectrometry.

Clopidogrel (ng/mL)Intraday accuracy (% of nominal concentration)Intraday precision (% of relative SD)Interday accuracy (% of nominal concentration)Interday precision (% of relative SD)
0.894.013.0101.013.0
4.5100.09.0103.010.0
11.0109.05.098.09.0
Table 2—

Mean ± SD values for pharmacokinetic parameters of clopidogrel and CAMD in horses after oral administration of a single dose of clopidogrel (2 mg/kg).

ParameterClopidogrel (n = 5)CAMD (n = 6)
Cmax (ng/mL)4.53 ± 3.3040.70 ± 23.00
tmax (h)0.54 ± 0.250.71 ± 0.19
λz (1/h)0.473 ± 0.7440.860 ± 0.359
t1/2 (h)1.81 ± 1.820.97 ± 0.49
AUC0–∞ (h•ng/mL)5.67 ± 2.4339.70 ± 15.80
AUCextrap (%)17.70 ± 13.404.54 ± 5.98

Parameters were determined by use of noncompartmental analysis.

AUCextrap = Percentage of the AUC that was extrapolated to infinity. AUC0–∞ = The AUC from time 0 to infinity. Cmax = Maximal plasma concentration. λz = Slope of the terminal portion of the plasma concentration curve. tmax = Time to maximal plasma concentration.

Of the 5 horses used to determine pharmacokinetic parameters, 1 was the horse with platelets that aggregated normally in response to 12.5μM ADP at all times after clopidogrel administration. Maximum clopidogrel concentration in this horse was 3.99 ng/mL, and the relative peak CAMD concentration was 88% of that of the other 4 horses. There were no significant linear relationships between any of the pharmacokinetic parameters for clopidogrel or CAMD and inhibition of platelet aggregation (data not shown).

One horse was not included for determination of pharmacokinetic parameters for clopidogrel. Platelets of that horse responded to clopidogrel, but the plasma concentration of clopidogrel was measurable only at a single time point after administration (0.53 ng/mL at 15 minutes after administration). Thus, pharmacokinetic parameters could not be derived for this horse and were not included in the subsequent analysis (Table 2). Concentrations of CAMD were detected in the plasma of this horse for up to 6 hours after clopidogrel administration; relative maximum CAMD concentration was 26% that of the other 5 horses. Pharmacokinetic data for CAMD derived for this horse were included in the subsequent analysis of pharmacokinetic parameters. Although clopidogrel is a weakly acidic drug (pKa = 4.55) and variation in equine gastric pH may affect absorption, peak clopidogrel or CAMD concentrations and gastric pH measured at the time of drug administration were not significantly associated (R2 = 0.29 and 0.13, respectively; Figure 3).

Discussion

Stimulation of activation of human platelets by ADP is mediated through 2 G-protein–coupled receptors, P2Y1 and P2Y12, which activate separate downstream responses. Stimulation of P2Y1 is linked to increased cytoplasmic calcium concentrations, shape changes, and transient aggregation.18,19 Stimulation of P2Y12 induces platelet degranulation, generation of thromboxane A2 that leads to autocrine stimulation, and inside-out upregulation of the fibrinogen receptor αIIb-βIIIa integrin that results in prolonged platelet aggregation.20–22 The P2Y12 receptor is thought to be the primary mediator of ADP-induced activation of equine platelets for 2 reasons.23 First, in contrast to human platelets, equine platelets do not undergo marked shape changes in response to ADP. Second, concentrations of the competitive P2Y12 inhibitor AR-C67085 that have no effect on human platelets will almost completely inhibit aggregation of equine platelets.

Although the underlying cause is unknown, clopidogrel is ineffective at inhibiting ADP-dependent aggregation of platelets in up to 33% of horses,24,25 15% of cats,10 and 56% of humans undergoing coronary stenting.26 One horse of the study reported here had platelets that were only weakly inhibited by clopidogrel at all times after administration. Poor responsiveness to clopidogrel in humans with defective CYP2C19 has been associated with reduced metabolism of the drug to the active metabolite.11 However, plasma concentrations of both clopidogrel and CAMD in the horse that responded poorly in the present study were comparable with those measured for the horses that responded to the drug. Poor outcomes for humans treated with clopidogrel have been attributed to polymorphisms in the gene encoding the P2Y12 receptor.27 Polymorphisms in this gene have also been identified in cats,28 and findings for the present study raised the possibility that such polymorphisms may exist in horses as well.

In humans, CAMD covalently binds to the P2Y12 receptor and blocks the associated ADP-induced signaling for the life of a platelet.11 In the study reported here, we found that doubling the ADP concentration to which platelets were exposed overcame the inhibition of the P2Y12 receptor during the first 24 hours after administration of clopidogrel to horses. Although local ADP concentrations have not been measured at sites of active thrombosis, strenuous exercise alone is sufficient to increase circulating ADP concentrations (2.5-fold increase) in humans.29 This concentration dependence is characteristic of reversible competitive inhibition, instead of the covalent noncompetitive inhibition of platelets that occurs with clopidogrel treatment of humans.

To directly assess effects of ADP on the equine platelet P2Y12 receptor, phosphorylation of the ADP-linked, intracellular-signaling protein VASP was evaluated. In humans30 and cats,10 VASP phosphorylation has been used to measure inhibition of platelet P2Y12 receptors after clopidogrel treatment. To eliminate contributions from clopidogrel or CAMD that was not covalently bound to P2Y12 receptors, washed platelets were used to measure VASP phosphorylation in the present study. To block contributions from the P2Y1 receptor pathway, platelets were stimulated with ADP in the presence of PGE1. For these conditions, relative VASP phosphorylation in equine platelets remained unchanged from preadministration values at all times after administration of clopidogrel. These results were consistent with the hypothesis that the equine P2Y12 receptor is inhibited in a competitive manner for the first 24 hours after treatment with clopidogrel. Consistent with these observations, clopidogrel reportedly has transient effects on equine platelets during the first 24 hours after administration.13 In contrast, administration of clopidogrel to humans results in a sustained decrease in VASP phosphorylation within 12 hours after administration,17,30 which is comparable to the inhibition of aggregation observed for human platelets.17

Absorption and elimination of clopidogrel and CAMD in horses were assessed to evaluate the relationship between circulating concentrations of these compounds and inhibition of equine platelets. For 1 horse of the study, clopidogrel was detectable at only a single time point; however, all horses rapidly absorbed the drug independent of gastric pH. Similar to results in humans, CAMD concentrations peaked within 1 hour after clopidogrel administration.11,31 In addition, CAMD reached a higher peak concentration in all horses than did clopidogrel, which indicated that clopidogrel underwent marked first-pass metabolism. These findings were consistent with previous measurements of the inactive clopidogrel metabolite SR 23664, which reaches a peak concentration within 30 minutes after oral administration of clopidogrel to horses.13

For all of the horses of the present study, substantial inhibition of platelet aggregation was evident 3 to 5 hours after concentrations of clopidogrel and CAMD peaked. A similar delay until onset of effects occurs in human platelets after oral administration of clopidogrel.32 The CAMD inhibits ADP-induced signaling by causing dispersion of multimeric P2Y12 receptor complexes out of lipid raft domains in platelet membranes.33 The delay in onset of drug action may be the result of this process because partitioning depends on both dimerization and diffusion rates of the receptors.34 This process may also have been involved in the extended action of the drug, which continued after concentrations of clopidogrel and CAMD had decreased to below the limit of quantitation.

The t1/2 values of clopidogrel and CAMD for horses were comparable to those measured for humans (6 and 0.5 hours, respectively). The mean life span of platelets is 6.6 to 9.2 days in horses, which suggests that approximately 10% to 20% of the platelet population is replaced every day.35 The continual emergence of an uninhibited platelet population may be an underlying cause of thrombotic events in treated humans.36 Newly formed reticulated platelets contribute overproportionately to the formation of aggregates in human patients undergoing long-term treatment with a combination of aspirin and thienopyridines, including clopidogrel. However, this effect does not occur in patients treated with aspirin and the direct-acting P2Y12 receptor inhibitor ticagrelor, which has a mean half-life of 7 hours.37 In combination with the observation that the antithrombotic effect of clopidogrel was reversible in horses for the first 24 hours after administration in the present study, these observations could indicate that effective administration of clopidogrel in horses may need to be more frequent than once a day.

Acknowledgments

Supported by funds provided by the Oak Tree Racing Association, the State of California Pari-Mutual Fund, and the Center for Equine Health through contributions by private donors. Funding sources did not have any involvement in the study design, data analysis and interpretation, or writing and publication of the manuscript.

The authors declare that there were no conflicts of interest.

ABBREVIATIONS

AUC

Area under the curve

CAMD

Clopidogrel active metabolite derivative

PG

Prostaglandin

PRP

Platelet-rich plasma

t1/2

Terminal-phase half-life

VASP

Vasodilator-stimulated phosphoprotein

Footnotes

a.

BD Vacutainer ACD-A tubes, Becton, Dickinson and Co, Franklin Lakes, NJ.

b.

Clopidogrel bisulfate, 75-mg tablets, Cipla Ltd, Malpur, India.

c.

AC•T Diff analyzer, Beckman-Coulter, Miami, Fla.

d.

Chrono-log 560-CA dual-channel aggregometer with Aggro/Link software, Chronolog, Havertown, Pa.

e.

Chronolog, Havertown, Pa.

f.

Calbiochem, San Diego, Calif.

g.

EMD Biosciences, Rockland, Mass.

h.

Thermo Scientific, Waltham, Mass.

i.

Bio-Rad Laboratories Inc, Hercules, Calif.

j.

p-VASP (16C2), Santa Cruz Biotechnology, Dallas, Tex.

k.

Goat anti-mouse IgG-HRP, Santa Cruz Biotechnology, Dallas, Tex.

l.

SuperSignal West Femto western blotting substrate, Thermo Scientific, Waltham, Mass.

m.

ChemiDoc-It system, UVP LLC, Upland, Calif.

n.

Sigma-Aldrich Corp, St Louis, Mo.

o.

Toronto Research Chemicals, Toronto, ON, Canada.

p.

LTQXL Orbitrap mass spectrometer, Thermo Scientific, San Jose, Calif.

q.

Acquity chromatography system, Waters Corp, Milford, Mass.

r.

ACE 3 C18 10-cm × 2.1-mm, 3-μm column, Mac-Mod Analytical Inc, Chadds Ford, Pa.

s.

Quan Browser, Xcalibur software, version 2.0.3.0, Thermo Fisher Scientific, San Jose, Calif.

t.

Phoenix, version 8.0, Certera, Princeton, NJ.

References

  • 1. Iba T, Levy JH. Inflammation and thrombosis: roles of neutrophils, platelets and endothelial cells and their interactions in thrombus formation during sepsis. J Thromb Haemost 2018;16:231241.

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  • 2. Weiss DJ, Geor RJ, Johnston G, et al. Microvascular thrombosis associated with onset of acute laminitis in ponies. Am J Vet Res 1994;55:606612.

    • Search Google Scholar
    • Export Citation
  • 3. Adam EN, Southwood LL. Primary and secondary limb cellulitis in horses: 44 cases (2000–2006). J Am Vet Med Assoc 2007;231:16961703.

  • 4. Fjordbakk CT, Arroyo LG, Hewson J. Retrospective study of the clinical features of limb cellulitis in 63 horses. Vet Rec 2008;162:233236.

  • 5. Weiss DJ, Trent AM, Johnston G. Prothrombotic events in the prodromal stages of acute laminitis in horses. Am J Vet Res 1995;56:986991.

    • Search Google Scholar
    • Export Citation
  • 6. Bailey SR, Adair HS, Reinemeyer CR, et al. Plasma concentrations of endotoxin and platelet activation in the developmental stage of oligofructose-induced laminitis. Vet Immunol Immunopathol 2009;129:167173.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 7. Bailey SR, Andrews MJ, Elliott J, et al. Actions and interactions of ADP, 5-HT, histamine and PAF on equine platelets. Res Vet Sci 2000;68:175180.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 8. Sanofi-Aventis. Product monograph: Plavix (clopidogrel bisulphate). Version 5. Paris: Sanofi-Aventis, 2009.

  • 9. Hogan DF, Fox PR, Jacob K, et al. Secondary prevention of cardiogenic arterial thromboembolism in the cat: the double-blind, randomized, positive-controlled feline arterial thromboembolism; clopidogrel vs. aspirin trial (FAT CAT). J Vet Cardiol 2015;17:S306S317.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 10. Li RH, Stern JA, Ho V, et al. Platelet activation and clopidogrel effects on ADP-induced platelet activation in cats with or without the A31P mutation in MYBPC3. J Vet Intern Med 2016;30:16191629.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 11. Wallentin L. P2Y12 inhibitors: differences in properties and mechanisms of action and potential consequences for clinical use. Eur Heart J 2009;30:19641977.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 12. Brainard BM, Kleine SA, Papich MG, et al. Pharmacodynamic and pharmacokinetic evaluation of clopidogrel and the carboxylic acid metabolite SR 26334 in healthy dogs. Am J Vet Res 2010;71:822830.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 13. Brainard BM, Epstein KL, LoBato D, et al. Effects of clopidogrel and aspirin on platelet aggregation, thromboxane production, and serotonin secretion in horses. J Vet Intern Med 2011;25:116122.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 14. Roscher KA, Failing K, Moritz A. Inhibition of platelet function with clopidogrel, as measured with a novel whole blood impedance aggregometer in horses. Vet J 2015;203:332336.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 15. Schwarz UR, Geiger J, Walter U, et al. Flow cytometry analysis of intracellular VASP phosphorylation for the assessment of activating and inhibitory signal transduction in human platelets: definition and detection of ticlopidine/clopidogrel effects. Thromb Haemost 1999;82:11451152.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 16. Takahashi M, Pang H, Kawabata K, et al. Quantitative determination of clopidogrel active metabolite in human plasma by LC-MS/MS. J Pharm Biomed Anal 2008;48:12191224.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 17. Geiger J, Teichmann L, Grossmann R, et al. Monitoring of clopidogrel action: comparison of methods. Clin Chem 2005;51:957965.

  • 18. Daniel JL, Dangelmaier C, Jin J, et al. Molecular basis for ADP-induced platelet activation. I. Evidence for three distinct ADP receptors on human platelets. J Biol Chem 1998;273:20242029.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 19. Jin J, Daniel JL, Kunapuli SP. Molecular basis for ADP-induced platelet activation. II. The P2Y1 receptor mediates ADP-induced intracellular calcium mobilization and shape change in platelets. J Biol Chem 1998;273:20302034.

    • Search Google Scholar
    • Export Citation
  • 20. Daniel JL, Dangelmaier C, Jin J, et al. Role of intracellular signaling events in ADP-induced platelet aggregation. Thromb Haemost 1999;82:13221326.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 21. Kauffenstein G, Bergmeier W, Eckly A, et al. The P2Y12 receptor induces platelet aggregation through weak activation of the αIIbβ3 integrin—a phosphoinositide 3-kinase-dependent mechanism. FEBS Lett 2001;505:281290.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 22. Paul BZS, Jin J, Kunapuli SP. Molecular mechanism of thromboxane A2-induced platelet aggregation. Essential role for P2Tac and α2a receptors. J Biol Chem 1999;274:2910829114.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 23. Mateos-Trigos G, Evans RJ, Heath MF. Effects of a P2Y12 receptor antagonist on the response of platelets to ADP. Comparison with human platelets. Res Vet Sci 2002;73:171175.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 24. Brooks MB, Divers TJ, Watts AE, et al. Effects of clopidogrel on the platelet activation response in horses. Am J Vet Res 2013;74:12121222.

  • 25. Watts AE, Ness SL, Divers TJ, et al. Effects of clopidogrel on horses with experimentally induced endotoxemia. Am J Vet Res 2014;75:760769.

  • 26. Serebruany VL, Steinhubl SR, Berger PB, et al. Variability in platelet responsiveness to clopidogrel among 544 individuals. J Am Coll Cardiol 2005;45:246251.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 27. Ziegler S, Schillinger M, Funk M, et al. Association of a functional polymorphism in the clopidogrel target receptor gene, P2Y12, and the risk for ischemic cerebrovascular events in patients with peripheral artery disease. Stroke 2005;36:13941399.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 28. Ueda Y, Li RHL, Tablin F, et al. Nonsynonymous single nucleotide polymorphisms in candidate genes P2RY1, P2RY12 and CYP2C19 for clopidogrel efficacy in cats. Anim Genet 2018;49:356357.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 29. Yegutkin GG, Samburski SS, Mortensen SP, et al. Intravascular ADP and soluble nucleotidases contribute to acute prothrombotic state during vigorous exercise in humans. J Physiol 2007;579:553564.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 30. Delavenne X, Mallouk N, Piot M, et al. Is there really a relationship between the plasma concentration of the active metabolite of clopidogrel and the results of platelet function tests? J Thromb Haemost 2010;8:23342338.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 31. von Beckerath N, Taubert D, Pogatsa-Murray G, et al. Absorption, metabolism, and antiplatelet effects of 300-, 600-, and 900-mg loading doses of clopidogrel: results of the ISAR-CHOICE (Intracoronary Stenting and Antithrombotic Regimen: Choose Between 3 High Oral Doses for Immediate Clopidogrel Effect) trial. Circulation 2005;112:29462950.

    • Search Google Scholar
    • Export Citation
  • 32. Montalescot G, Sideris G, Meulemann C, et al. A randomized comparison of high clopidogrel loading doses in patients with non-ST-segment elevation acute coronary artery syndromes: the ALBION (Assessment of the Best Loading Dose of Clopidogrel to Blunt Platelet Activation, Inflammation and Ongoing Necrosis) trial. J Am Coll Cardiol 2006;48:931938.

    • Search Google Scholar
    • Export Citation
  • 33. Savi P, Zachayus JL, Delesque-Touchard N, et al. The active metabolite of clopidogrel disrupts P2Y12 receptor oligomers and partitions them out of lipid rafts. Proc Natl Acad Sci U S A 2006;103:1106911074.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 34. Fallahi-Sichani M, Linderman JJ. Lipid raft mediated regulation of g-protein coupled receptor signaling by ligands which influence receptor dimerization: a computational study. PLoS One 2009;4:e6604.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 35. Carter EI, Valli VE, McSherry BJ, et al. The kinetics of hematopoiesis in the light horse I. The lifespan of peripheral blood cells in the normal horse. Can J Comp Med 1974;38:303313.

    • Search Google Scholar
    • Export Citation
  • 36. Armstrong PC, Hoefer T, Knowles RB, et al. Newly formed reticulated platelets undermine pharmacokinetically short-lived antiplatelet therapies. Arterioscler Thromb Vasc Biol 2017;37:949956.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 37. AstraZeneca. Product monograph: Brilinta (ticagrelor). Cambridge, England: AstraZeneca, 2016.

  • Figure 1—

    Aggregation response of equine platelets to ADP before (time 0) and after oral administration of a single dose of clopidogrel (2 mg/kg). A—Representative absorption curves for PRP in response to 12.5μM ADP. Samples were collected before (solid black line) and 2 (dashed black line), 4 (dotted black line), 6 (solid gray line), 12 (dashed gray line), and 24 (dotted gray line) hours after clopidogrel administration. Time of ADP addition is indicated (arrow). B—Minimum relative absorption of PRP after addition of 12.5μM ADP. Samples were obtained from 6 horses before and 12 hours after administration of clopidogrel. Each symbol represents values for 1 horse; the white symbols represent values for a horse with platelets that responded normally to ADP at all times after drug administration. C—Minimum relative absorption of PRP in response to 12.5μM ADP for all samples obtained from 5 horses in which platelet aggregation was inhibited by clopidogrel. Each symbol represents results for 1 horse, and the mean for all 5 horses at each time is indicated (horizontal line). *Platelet aggregation was significantly (P < 0.05) inhibited, compared with preadministration values. D—Comparison of minimum relative absorption for equine PRP in response to 12.5μM ADP (circles) or 25μM ADP (squares) before and after administration of clopidogrel. Values represent mean ± SD for 5 horses. ‡Within a time point, value differs significantly (P < 0.01) for stimulation with 12.5μM ADP from the value for simulation with 25μM ADP.

  • Figure 2—

    Results for VASP phosphorylation of equine platelets after oral administration of a single dose of clopidogrel. A—Representative western blots of VASP phosphorylation in washed platelets obtained from horses before (time 0) and at various times after oral administration of clopidogrel. Platelets were incubated with 20μM ADP (lane A), 3.3μM PGE1 (lane P), or both 20μM ADP and 3.3μM PGE1 (lane B). Equal numbers of platelets were loaded in each lane. B—Mean ± SD values for the platelet reactivity index (PRI) determined from relative VASP phosphorylation for 5 horses with substantial inhibition of platelet aggregation in response to clopidogrel. The PRI is a variable without units.

  • Figure 3—

    Mean ± SD plasma concentrations of clopidogrel (circles and solid line) and CAMD (squares and dashed line) before (time 0) and after oral administration of a single dose of clopidogrel to horses (n = 5; A), and peak plasma concentrations of clopidogrel (circles) and CAMD (squares) as a function of gastric pH in 6 horses (B). Clopidogrel and CAMD concentrations were estimated on the basis of the clopidogrel calibration curve. In panel A, time until maximum plasma concentrations of clopidogrel and CAMD was detected ranged from 15 to 45 minutes and 30 to 60 minutes, respectively.

  • 1. Iba T, Levy JH. Inflammation and thrombosis: roles of neutrophils, platelets and endothelial cells and their interactions in thrombus formation during sepsis. J Thromb Haemost 2018;16:231241.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 2. Weiss DJ, Geor RJ, Johnston G, et al. Microvascular thrombosis associated with onset of acute laminitis in ponies. Am J Vet Res 1994;55:606612.

    • Search Google Scholar
    • Export Citation
  • 3. Adam EN, Southwood LL. Primary and secondary limb cellulitis in horses: 44 cases (2000–2006). J Am Vet Med Assoc 2007;231:16961703.

  • 4. Fjordbakk CT, Arroyo LG, Hewson J. Retrospective study of the clinical features of limb cellulitis in 63 horses. Vet Rec 2008;162:233236.

  • 5. Weiss DJ, Trent AM, Johnston G. Prothrombotic events in the prodromal stages of acute laminitis in horses. Am J Vet Res 1995;56:986991.

    • Search Google Scholar
    • Export Citation
  • 6. Bailey SR, Adair HS, Reinemeyer CR, et al. Plasma concentrations of endotoxin and platelet activation in the developmental stage of oligofructose-induced laminitis. Vet Immunol Immunopathol 2009;129:167173.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 7. Bailey SR, Andrews MJ, Elliott J, et al. Actions and interactions of ADP, 5-HT, histamine and PAF on equine platelets. Res Vet Sci 2000;68:175180.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 8. Sanofi-Aventis. Product monograph: Plavix (clopidogrel bisulphate). Version 5. Paris: Sanofi-Aventis, 2009.

  • 9. Hogan DF, Fox PR, Jacob K, et al. Secondary prevention of cardiogenic arterial thromboembolism in the cat: the double-blind, randomized, positive-controlled feline arterial thromboembolism; clopidogrel vs. aspirin trial (FAT CAT). J Vet Cardiol 2015;17:S306S317.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 10. Li RH, Stern JA, Ho V, et al. Platelet activation and clopidogrel effects on ADP-induced platelet activation in cats with or without the A31P mutation in MYBPC3. J Vet Intern Med 2016;30:16191629.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 11. Wallentin L. P2Y12 inhibitors: differences in properties and mechanisms of action and potential consequences for clinical use. Eur Heart J 2009;30:19641977.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 12. Brainard BM, Kleine SA, Papich MG, et al. Pharmacodynamic and pharmacokinetic evaluation of clopidogrel and the carboxylic acid metabolite SR 26334 in healthy dogs. Am J Vet Res 2010;71:822830.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 13. Brainard BM, Epstein KL, LoBato D, et al. Effects of clopidogrel and aspirin on platelet aggregation, thromboxane production, and serotonin secretion in horses. J Vet Intern Med 2011;25:116122.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 14. Roscher KA, Failing K, Moritz A. Inhibition of platelet function with clopidogrel, as measured with a novel whole blood impedance aggregometer in horses. Vet J 2015;203:332336.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 15. Schwarz UR, Geiger J, Walter U, et al. Flow cytometry analysis of intracellular VASP phosphorylation for the assessment of activating and inhibitory signal transduction in human platelets: definition and detection of ticlopidine/clopidogrel effects. Thromb Haemost 1999;82:11451152.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 16. Takahashi M, Pang H, Kawabata K, et al. Quantitative determination of clopidogrel active metabolite in human plasma by LC-MS/MS. J Pharm Biomed Anal 2008;48:12191224.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 17. Geiger J, Teichmann L, Grossmann R, et al. Monitoring of clopidogrel action: comparison of methods. Clin Chem 2005;51:957965.

  • 18. Daniel JL, Dangelmaier C, Jin J, et al. Molecular basis for ADP-induced platelet activation. I. Evidence for three distinct ADP receptors on human platelets. J Biol Chem 1998;273:20242029.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 19. Jin J, Daniel JL, Kunapuli SP. Molecular basis for ADP-induced platelet activation. II. The P2Y1 receptor mediates ADP-induced intracellular calcium mobilization and shape change in platelets. J Biol Chem 1998;273:20302034.

    • Search Google Scholar
    • Export Citation
  • 20. Daniel JL, Dangelmaier C, Jin J, et al. Role of intracellular signaling events in ADP-induced platelet aggregation. Thromb Haemost 1999;82:13221326.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 21. Kauffenstein G, Bergmeier W, Eckly A, et al. The P2Y12 receptor induces platelet aggregation through weak activation of the αIIbβ3 integrin—a phosphoinositide 3-kinase-dependent mechanism. FEBS Lett 2001;505:281290.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 22. Paul BZS, Jin J, Kunapuli SP. Molecular mechanism of thromboxane A2-induced platelet aggregation. Essential role for P2Tac and α2a receptors. J Biol Chem 1999;274:2910829114.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 23. Mateos-Trigos G, Evans RJ, Heath MF. Effects of a P2Y12 receptor antagonist on the response of platelets to ADP. Comparison with human platelets. Res Vet Sci 2002;73:171175.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 24. Brooks MB, Divers TJ, Watts AE, et al. Effects of clopidogrel on the platelet activation response in horses. Am J Vet Res 2013;74:12121222.

  • 25. Watts AE, Ness SL, Divers TJ, et al. Effects of clopidogrel on horses with experimentally induced endotoxemia. Am J Vet Res 2014;75:760769.

  • 26. Serebruany VL, Steinhubl SR, Berger PB, et al. Variability in platelet responsiveness to clopidogrel among 544 individuals. J Am Coll Cardiol 2005;45:246251.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 27. Ziegler S, Schillinger M, Funk M, et al. Association of a functional polymorphism in the clopidogrel target receptor gene, P2Y12, and the risk for ischemic cerebrovascular events in patients with peripheral artery disease. Stroke 2005;36:13941399.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 28. Ueda Y, Li RHL, Tablin F, et al. Nonsynonymous single nucleotide polymorphisms in candidate genes P2RY1, P2RY12 and CYP2C19 for clopidogrel efficacy in cats. Anim Genet 2018;49:356357.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 29. Yegutkin GG, Samburski SS, Mortensen SP, et al. Intravascular ADP and soluble nucleotidases contribute to acute prothrombotic state during vigorous exercise in humans. J Physiol 2007;579:553564.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 30. Delavenne X, Mallouk N, Piot M, et al. Is there really a relationship between the plasma concentration of the active metabolite of clopidogrel and the results of platelet function tests? J Thromb Haemost 2010;8:23342338.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 31. von Beckerath N, Taubert D, Pogatsa-Murray G, et al. Absorption, metabolism, and antiplatelet effects of 300-, 600-, and 900-mg loading doses of clopidogrel: results of the ISAR-CHOICE (Intracoronary Stenting and Antithrombotic Regimen: Choose Between 3 High Oral Doses for Immediate Clopidogrel Effect) trial. Circulation 2005;112:29462950.

    • Search Google Scholar
    • Export Citation
  • 32. Montalescot G, Sideris G, Meulemann C, et al. A randomized comparison of high clopidogrel loading doses in patients with non-ST-segment elevation acute coronary artery syndromes: the ALBION (Assessment of the Best Loading Dose of Clopidogrel to Blunt Platelet Activation, Inflammation and Ongoing Necrosis) trial. J Am Coll Cardiol 2006;48:931938.

    • Search Google Scholar
    • Export Citation
  • 33. Savi P, Zachayus JL, Delesque-Touchard N, et al. The active metabolite of clopidogrel disrupts P2Y12 receptor oligomers and partitions them out of lipid rafts. Proc Natl Acad Sci U S A 2006;103:1106911074.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 34. Fallahi-Sichani M, Linderman JJ. Lipid raft mediated regulation of g-protein coupled receptor signaling by ligands which influence receptor dimerization: a computational study. PLoS One 2009;4:e6604.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 35. Carter EI, Valli VE, McSherry BJ, et al. The kinetics of hematopoiesis in the light horse I. The lifespan of peripheral blood cells in the normal horse. Can J Comp Med 1974;38:303313.

    • Search Google Scholar
    • Export Citation
  • 36. Armstrong PC, Hoefer T, Knowles RB, et al. Newly formed reticulated platelets undermine pharmacokinetically short-lived antiplatelet therapies. Arterioscler Thromb Vasc Biol 2017;37:949956.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 37. AstraZeneca. Product monograph: Brilinta (ticagrelor). Cambridge, England: AstraZeneca, 2016.

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